The term meningitis is a general one, referring to the inflammatory response to infection of the meninges and the cerebrospinal fluid (CSF). See Roos, "Chapter 16", in Scheld, et al. eds., 1991, Infections of the Central Nervous System:335-403 which is incorporated herein in its entirety by reference.
The fact that the inflammatory response occurs in the proximity of the brain and in the space limited by a rigid cranium, makes these infections serious and life threatening. Most patients exhibit nonspecific clinical signs and symptoms such as fever, irritability, altered mental status usually accompanied by vomiting and loss of appetite. In children one year of age and older, photophobia and headache are common complaints. Specific clinical signs indicative of meningitis are neck rigidity and pain on neck flexicn. Brudzinski's sign (neck flexion producing knee and hip flexicn) and Kernig's sign (difficulty and pain in raising extended leg) are other useful clinical signs.
In infants less than 6 months old, early diagnosis of meningitis is difficult because signs of meningitis are not prominent and neck rigidity is often absent. Such patients commonly exhibit fever, respiratory distress, other signs of sepsis, and convulsions. Bulging anterior fontanelle due to increased intracranial pressure may be the only specific sign.
Petechiae (or rash) is most commonly present in meningococcal infections. In severe meningococcal infections with bacteraemia, petechiae and shock may develop with alarming rapidity. Convulsions at some point in the illness occur in about 30% of the cases. This number is often higher in neonares and infants under one year of age. Other acute complications include septic shock, disseminated intravascular coagulation, syndrome of inappropriate antidiuretic hormone, increased intracranial pressure, and diabetes insipidus. Convulsions and coma appearing with 24 hours accompanied by high fever indicates serious infection (Stutman & Marks, 1987, Clin. Ped. 26:432-438).
A diverse array of both bacteria and viruses cause meningitis, the infectivity of which is dependent on a complex array of factors, including virulence of the organisms, the carrier state, and the host's humoral immune response.
Viruses generally cause milder forms of meningitis (eg. meningomyelitis and aseptic meningitis) with a short clinical course and reduced mortality. Agents most commonly associated are coxsackievirus A (types 2,4,7,9,10), B (types 1-6), polio virus, echoviruses (types 1-34, except 12,24,26,29,32-34), enteroviruses (types 70, 71), human immunodeficiency virus-1 (HIV-1), and rubella virus (RV). See Melnick, "Chapter 33" and Cooper, "Chapter 42" in Fields, et al., eds., 1985, Virology: 739-794 and 1005-1032, respectively; and Rotbart, "Chapter 3", in Scheld et al., 1991, infra:19-33 which are all incorporated herein by reference.
Rubella is possibly the most common cause of viral meningitis. Moreover, the most common chemical sequelae of rubella infection of young children are meningitis, meningomyelitis and rubella associated panencephalitis. Rubella is a highly contagious disease, usually associated with childhood, and is characterized by a general rash and a mild fever. Sub-clinical infections are also common. Its clinical aspects have been confused with measles, which it closely resembles. Since its early discovery in Germany, Rubella is often referred to as German measles. The infection of a pregnant woman poses the greatest risk when infection of the fetus can lead to spontaneous abortion or an array of abnormalities called the Congenital Rubella Syndrome in the newborn. Damage most frequently involves cardiac abnormalities, deafness, cataracts, blindness and Central Nervous System (CNS) disorders including microencephaly.
The rubella virion is a spherical, enveloped virus, approximately 60 nm in diameter, and is a member of the Togaviridae. It's genome is a 10 Kb plus single-stranded RNA. The outer envelope is comprised of lipoproteins derived from the infected host cell, and it appears to have two viral encoded glycoproteins, E1 (58 Kd) and E2 (42-47 Kd), responsible for the hemagglutination activity of the virus. Its core protein is a non-glycosylated nucleocapsid protein with an approximate weight of 33 Kd. It appears that the core, E1, and E2 are all derived from the same parent protein--Structural Polyprotein. See Clark et al., 1987, Nucl. Acids Res. 15:3041-3057; Dominguez, et al., 1990, Virolcgy 177:225-238, both which are incorporated herein by reference. Three strains of wild type RV (M33, Therien, Judith) and a vaccine strain (HPV77) of RV have been identified and sequenced (Zheng et alo, 1988, Arch. Virol. 98:189-197 incorporated herein in its entirety by reference). Between these different wild types strains, there exists minor variations in the amino acid sequence of the Structural Polyprotein (Dominguez, infra; Clarke, infra).
The detection of RV in diagnosis has in the past proven difficult, largely because the virus grows to low titers in the tissue cultures and is highly liable, making it technically difficult to isolate and purify (Ho-Terry et al., 1986, Arch. Virol. 87: 219-228).
The detection of RV in the CNS presents additional technical problems. It has been known since 1941 that the RV can infect cells of the CNS (Gregg, 1941, Trans. Ophthalmol. Soc. Aust. 3:35-46). However, it has proven difficult to reliably demonstrate the presence of the RV in infected brain tissue. Persistent infection of the CNS has been well documented in the congenital rubella syndrome (Desmond et al., 1967, J. Pediat., 7:311-331), and in the neuropathology if progressive rubella panencephalitis of late onset occurs where the virus has been isolated from brain biopsy material (Townsend et al., 1975, N. Engl. J. Med. 292:990-993; Cremer et al., 1979, J. Gen. Virol. 29:143-153). Less commonly documented are the wide range of neuropathies known to follow exposure to the RV. These include encephalitis, meningomyelitis, and bilateral optic neuritis (Connolly et al., 1975, Brain 98:583-594). Moreover, the report of a diffuse myelitis following RV in cells of the nervous system requires further investigation (Holt et al., 1975, Brit. Med. J., 7:1037-1038).
RV-directed polypeptide synthesis in normal rat glial cells in continuous tissue culture has been studied (Singh & Van Alstyne, 1978, Brain Res. 155:418-421). Unlike a productive rubella virus infection in permissive murine L (muscle) cells, infection of normal glial cells resulted in no detectable progeny virons in tissue culture supernatants and no detectable rubella 33 Kd core protein in infected cell lysates (Pope and Van Alstyne, 1981, Virology 124:173-180). Furthermore, exposure of infected gila to dibutyryl cyclic adenine monophosphate reversed the restriction, resulting in the appearance of the 33 Kd rubella nucleocapsid protein in infected cell lysates and the appearance of mature progeny virions in tissue culture supernatants (Van Alstyne and Paty, 1983, Virology 124:173-180).
Others have reported a lack of synthesis of the structural M protein in measles virus-infected brain cells obtained from subacute sclerosing panencephalitis autopsy material established in tissue culture (Hall and Choppin, 1979, Virology 99:443-447). Also, it is known that the incomplete synthesis of some Herpes specific structural proteins occurs during a nonpermissive infection of some cells of nervous system origin (Adler et al., 1978, J. Gen. Virology 39:9-20).
Taken together, these data indicate that even very different viruses may undergo restricted replication in brain cells. The synthesis of a limited number of viral gene products could account for incomplete virion assembly, the translation of polypeptides of variable molecular weights, alterations in the immune response to input virus, and difficulties in successful virus isolation from infected brain tissue.
Therefore, there remains a need for a diagnostic system which would detect RV protein antigens in CNS tissue in both the presence as well as the absence of an active, productive infection.
Early diagnostic tests were based on the hemagglutinating properties of its external glycoproteins. Commonly, the hemagglutination inhibition assays relied on the presence of antibodies to the RV hemagglutinin (HA) in the serum samples to inhibit the vital-mediated hemagglutination of chick red blood cells (Herrmann, "Rubella Virus", 1979, in Diagnostic Procedures For Vital, Rickettsial And Chlamydial Infections:725-766). The presence of high inhibition, indicated the indirect measurement of antibodies to the HA protein, and thereby, a recent rubella infection.
More recent tests employ enzyme-labelled antibodies in the enzyme-linked-immunosorbent assays (ELISA) (Voller & Bidwell, 1975, Br. J. Exp. Pathol. 56:338-339 incorporated herein by reference). These assays are also indirect tests to measure the amount of circulating antibody to RV as an indication of infection. Indirect ELISA tests for RV employ bound viral antigens on a plastic microwells and the presence of bound antibodies linked to enzymes such as horseradish peroxidase.
There are several problems with the use of the indirect RV ELISA kits. These relate to low antibody titers observed with RV infection, the need for elaborate "cut-off" value calculations to eliminate background binding, the limited use of the test in the detection of low levels of specific viral antigens present in chronic CNS infection, and the tedious and time consuming nature of the test performance.
A different use of monoclonal antibodies and their corresponding synthetic peptide epitopes may prove more useful in detecting RV infection in the CNS. There has been discussion that refers to the use of three non-competing monoclonal antibodies directed against the E1 glycoprotein, but this system has not been applied to CNS-specific diagnostics (Terry et al., 1988, Arch. Virol. 98, 189-197 incorporated herein by reference).
Therefore, there is clearly a need for a rapid and a sensitive diagnostic test for the detection of the RV in CNS infection.
Furthermore, a live, attenuated rubella vaccine has been developed (Parkman et al., 1966, N. Engl. J. Med 275: 569-574). This vaccine is immunogenic in at least 95% of the recipients, and does confer protection against reinfection, in spite of the fact that it induces antibody levels which are significantly lower than those generated by wild type virus infection. However, a serious drawback associated with the administration of the attenuated vaccine is the significant proportion of adult females that go on to develop rubella-associated arthritis. Furthermore, recently immunized individuals still harbour infectious virus and are therefore infectious, proving dangerous to pregnant women with whom they may be in contact.
Therefore, there is also a need for a non-infectious, innocuous vaccine. Such a vaccine could possibly be constructed from synthetic or recombinant peptides of RV proteins. Moreover, no epitope has yet been identified which would induce only neutralizing antibodies, necessary for conferring effective vaccine protection.
Another virus responsible for meningitis is the Human Immunodeficency Virus-1 (HIV-1). HIV-1 is a human retrovirus which has been identified as the etiological agent of AIDS, an infectious and fatal disease transmitted through intimate sexual contact and exposure to contaminated blood or blood products. HIV-1 is related to the lentiviruses on the basis of its biological and in vitro characteristics, morphology and nucleotide sequences. It is also referred to as Human T-cell Lymphotrophic Virus type III, Lymphadenopathy Associated Virus, and AIDS Associated Retrovirus (Gallo, et al., 1984, Science, 224:500-503; Sarngadharan, et al., 1984, Science, 224:506-508; Barre-Sinoussi, et al., 1983, Science, 220:868-871; Levy, 1984, Science, 225:840-842; Gonda et al., 1985 Science, 227:177-179; Stephan, et al., 1986, Science, 231:589-594). Much interest has been focused on the effect of the long term, persistent infection of the immune system, by HIV-1. Recent information indicates that the virus moves from blood to the lymph nodes and thymus where it remains active, culminating in viremia, a precipitous drop in the CD4+T-cell count, and one or more of the several symptoms known as AIDS.
However, primary HIV-1 infection itself results in an immediate set of defined clinical features. Commonly, an acute febrile illness resembling influenza or mononucleosis is noted. In addition, lymphocytic meningitis may accompany the febrile illness and the patient may then be presented with headache, stiff neck and photophobia, as well as rigors, arthralgias and myalgias, truncal maculcpapular rash, urticaria, abdominal cramps and diarrhea (Ho, 1985, Ann. Internal Medicine 103:880-883).
While some patients remain asymptomatic for up to 3 months preceding their seroconversion, indicating that HIV-1 infection may be subclinical, primary infection should be included in the differential diagnosis of prolonged febrile illnesses in persons at risk for AIDS. The presence of a maculopapular or urticarial rash, or lymphocytic meningitis is compatible with this diagnosis. Hence, early recognition of the varied syndromes associated with this virus might permit effective treatment before immunologic abnormalities become established.
There is, therefore, the need for a rapid, direct diagnostic test for viral meningitis, prior to seroconversion, when the transient meningitis may represent the initiation of a more serious, long term HIV-1 related illness.
Currently, one of the most commonly used direct tests for HIV-1 infection employs the following approaches: (i) direct culturing of virus from infected blood or,blood cells and subsequent in vitro propagation of the virus in lymphocyte cultures; (ii) measuring reverse transcriptase levels; (iii) immunocytochemical staining of viral proteins; (iv) electron microscopy; (v) hybridization of nucleic acid probes; and measuring HIV-1 antigens with enzyme immunoassays (Goudsmit et al., 1986, Brit. Med. J., 2993:1459-1462; Caruso et al., 1987, J. Virol. Methods, 17:199-210).
The HIV-1 appears to have at least three core protein (p17, p24, and p15) that are derived from a core polyprotein called gag polyprotein. See Muesing, et al., 1985, Nature 313:450-458 incorporated herein by reference. The gag polyprotein in the LV isolate of HIV-1 is 478 amino acids long and the three mature core proteins appear to be derived as p17 from amino acid sequence numbers 1-132, p24 from amino acid sequence numbers 133-391, and p15 from amino acid sequence numbers 392-478 (Muesing, infra). Moreover, it appears that the HIV-1 (LAV-la isolate) also has at least one capsid transmembrane glycoprotein derived from a 861 amino acid long Envelope Polyprotein (Wain-Hobson, et al., 1985, Cell 40:9-17 incorporated herein by reference).
The enzyme immunoassays have clearly shown the diagnostic importance of the presence of the p24 core protein. A correlation has been established between viremia, the decline of antibodies to p24, and the progression of symptoms from the asymptomatic seropositivity to fully expressed AIDS (Lange et al., 1986, Brit. Med. J., 293:1459-1462; Paul et al., 1987, J. Med. Virol., 22:357-363; Forster et al., 1987, AIDS, 1:235-240). A decline in the p24 level has also been observed to occur in patients treated with AZT (Chaisson et al., 1986, New Eng. J. Med., 315:1610-1611).
Assays for the direct detection of p24 are currently on the market (Allain, infra; Forster, infra). These assays use the same sandwich format in which serum samples are incubated with bound and enzyme-labelled anti-p24-antibodies to form an antibody/p24-antigen-antibody sandwich. Antigen levels of approximately 50 picograms/ml can be detected, when the antigen concentration is read from a standard curve constructed with a set of p24 standards of known concentrations. The tests are tedious and time consuming to perform, require dilutions of patients' sera, and do not provide information regarding the comparisons of rising antigen and concomitant declining antibody levels necessary to evaluate laboratory findings.
Therefore, the need to rapidly and effective diagnostic test to screen large numbers of a symptomatic individuals for the presence of HIV-1 virus in individuals is clear.
There is also an urgent need for a vaccine to afford protection against transmission of AIDS by individuals who are not detected by current diagnostic tests.
However, there are significant difficulties inherent in designing a vaccine which will confer protection against HIV-1. The vaccine must differentiate between HIV-1 and the closely-related virus, HIV-2. The rapid rate of HIV-1 mutation requires that the antigen(s) be highly conserved. Moreover, the HIV-1 infection of a small subset of T cells requires the killing of an integral part of the immune cell network, with unknown consequences, to completely eradicate the virus. In addition, vaccinated antigens could enter lymph nodes and stimulate B cells to produce cytokines that in turn stimulate HIV-1 infection of T cells, and thereby having a reverse effect, causing a more rapid onset of AIDS.
Peptides from gp120, gp160, gp41, gp120 +gp41, p17 and p14 are currently being employed for vaccine production by several companies and universities (Spalding, 1992, Biotech. 10:24-29.) However, these peptides are being tested for their ability to solely induce B cells to produce neutralizing antibody.
Therefore, there is an urgent need for the selection of HIV-1 peptides which would serve as appropriate B cell stimulators, to produce protective, neutralizing antibody, as well as appropriate cytokine blockers to prevent HIV-1 infection of T-cells. To date, no known combination of such peptides has been shown to protect against AIDS infection.
Bacteria are the other major cause of meningitis. Approximately 70% of all cases of bacterial meningitis occur in children under the age of 5 years; three bacterial species cause 84% of all meningitis cases reported in the United States: Haemophilus influenza type B, and Streptococcus pneumoniae and Neisseria meningitidis (Roos, infra; Stutman, infra). Less prevalent bacterial species include Pseudomonas aerugensosa, Staphylococci, Mycobacteria and Listeria species.
All strains of Haemophilus influenzae (H. influenza) are divided into two groups: typeable strains which commonly have a capsule, and nontypeable strains which do not. Typing of the encapsulated strains is accomplished by serological techniques, using reference antisera. Types a to f have been identified in this way. Those strains which fail to react with any of the reference antisera are classified as nontypeable.
The most frequent cause of neonatal meningitis and other invasive infections in the United States is the encapsulated H. influenzae type b (Hib) (Fraser et al., 1974, Am. J. Epidemiol,. 100:29-34). While the major incidence of childhood meningitis occurs between the ages of one and five years, 60% of the meningitis cases due to Hib occur in children under the age of two years.
The nontypeable H. influenzae are known to cause meningitis, pneumonia, bacteraemia, postpartum sepsis, and acute febrile tracheobronchitis in adults (Murphy et al., 1985, J. Infect. Diseases, 152:1300-1307). About 20 to 40% of all cases of otitis media are caused by this H. influenzae, which is a frequent etiologic agent of otitis media in children and young adults. Since infection confers no long lasting immunity, repeated infections of the same organism is frequently observed. These chronic ototis media infections are treated by administration of antibiotics, and drainage of the inner ear, where such a procedure is deemed necessary. H. influenzae strains have also been implicated as a primary cause of sinusitis (Cherry & Dudley, 1981, in Feigin & Cherry eds., Textbook of Pediatric Infectious Diseases:103-105). Nontypeable H. influenzae are also known to cause neonatal sepsis.
A vaccine is currently available for protection against typeable influenzae, and employs the capsular polysaccharide antigen of Hib, polyribosyl ribitol phosphate (Smith et al., 1973, Pediatrics, 52:637-644; Anderson et al., 1972, J. Clin. Inv., 51:31-88). However, Anti-PRP antibody is not effective in conferring protection against non-typeable H. influenzae infection. Thus, all available vaccines against H. influenzae are all directed against Hib, and all elicit anti-PRP antibody to confer protection. Since the non-typeable H. influenzae lack the PRP capsule, no vaccine is efficacious against this group.
However, there does appears that H. Influenzae exhibits an outer membrane lipoprotein referred to as p4 (Green, et al., 1992, EMBL Bank, incorporated herein by reference). The p4 protein appears to be derived from the Lipoprotein E Precursor, the precursor protein being 274 amino acids in length. (SEQ ID NO: 17)
There is therefore a clear need for both a method of diagnosis for this disease as well as a vaccine which would protect against both typeable as well as nontypeable H. influenzae. It is possible that the p4 lipoprotein providing a source for such a vaccine.
Streptococcus pneumoniae is the leading cause of community-acquired bacterial pneumonia (pneumococcal diseases), with approximately 500,000 cases a year reported in the United States. Bacterial pneumonia is most prevalent among the very young, the elderly and immuno-compromised persons. In infants and children, pneumococci are the most common bacterial cause of pneumonia, otitis media and bacteraemia and a less common cause of meningitis (causing 20-25% of reported cases).
Pneumococci are carried in the respiratory tract of a significant number of healthy individuals. But, in spite of the high carriage rate, its presence does not necessarily imply infection. However, if one of the highly pathogenic pneumococcal types, such as S. pneumoniae, is isolated from rusty-colored sputum (also containing a large number of polymorphonuclear leucocytes), body fluids, blood cultures, or specimens collected via transtracheal or lung puncture from the lower respiratory tract, its detection is usually significant.
S. pneumoniae is a gram positive bacteria. Proteins located on the cell surface of many gram positive bacteria are frequently involved in virulence and host immunity and have, in the past, been used in typing these bacteria and in immunoprotection studies. There are a large number of S. pneumoniae strains, classified into serotypes based on their surface carbohydrate structures. There are also many cell surface proteins associated with S. pneumoniae. Surface proteins that exhibit antigenic variation (by antigenic shirt or drift) make the identification of a common but exclusive cell surface antigen difficult and may provide the organism with an additional mechanism for evading the host immune response.
Detection of this bacteria at an early stage is essential to facilitate treatment of the infection. Thus, it is important to be able to quickly identify whether S. pneumoniae is present in a patient and to be able to follow the effect of antibiotic treatment on the bacteria. As available immunoassays for S. pneumoniae antigen detection are deficient for lack of specificity and/or sensitivity, there remains the need for an improved method of such detection.
Monoclonal antibody (Mab) technology has recently provided researchers with tools to reproducibly and accurately analyze the cell surface components of S. pneumoniae. Hence S. pneumoniae proteins are of interest to epidemiologists as they may provide a method of detection as well as for vaccines against the bacteria.
One such cell surface protein is Streptococcus pneumoniae pneumonococcal surface protein A (pspA) (Yother, 1992, J. Bacteriol. 174:601-609 incorporated herein by reference). The complete sequence of this protein is known.
It is known that one such pneumonococcal vaccine has been developed which incorporates the capsular polysaccharide antigens of 23 prevalent serotypes of pneumococci. These serotypes are responsible for 87% of pneumococcal disease in the United States. This second generation vaccine replaced a 14-valent polysaccharide vaccine available since 1977. However, the U.S. Department of Health and Human Services has stated that a more immunogenic pneumococcal vaccine is needed, particularly for children younger than 2 years of age. This necessity exists because the 23-valent vaccine is poorly immunogenic in this age group. Consequently, the use of the vaccine is not recommended in children with recurrent upper respiratory diseases, such as otitis media and sinusitis. Furthermore, the 23-valent vaccine is only 44-61% efficacious when administered to persons over 65 years old, and revaccination is not advised. Thus, there remains a clear need for an improved pneumococcal vaccine.
Neisseria meningitis (N. Meningitis) is one of the leading causes of community-acquired bacterial meningitis, causing 10.3% of cases in the United States between 1978-1981 (Tunkel et al., 1990 Annals of Internal Medicine, 112: 610-623). Meningococcal meningitis is most prevalent among infants between 6-12 months and adolescents (Larter & Master, 1992, Am. J. Med.--Infectious Disease Symposium:120-123). In addition to meningococemia, other less commonly associated diseases such as conjunctivitis, sinusitis, endocarditis, and primary pneumonia can occur (Duerden, 1988, J. Med. Microbiol., 26:161-187).
N. meningitidis bacterium are carried in the nasopharynx of 10-15% of healthy individuals. In spite of the high carriage rate, its presence does not necessarily imply infection. However, isolation of N. meningitidis from cerebral spinal fluid or blood culture is significant (Stutman, infra; Mendelson & Dascal, 1992, Can. J. of Diag., 9:47-57; Martin, 1983, Am. J. Med.:120-123).
N. meningitidis is a gram negative bacteria. Proteins located on the cell surface of many gram negative bacteria have, in the past, been used in typing and immunoprotective studies. There are a large number of N. meningtidis strains and there are many cell surface proteins associated with N. meningtidis. This has made identification of a common but exclusive cell surface antigen difficult.
Detection of this bacteria at an early stage is essential to facilitate treatment of the infection (Stutman, infra). Thus, it is important to possess the ability to identify whether N. meningtidis is present in a patient and to follow the effect of antibiotic treatment on the bacteria. As available immunoassays for N. meningtidis antigen detection have shown lack of specificity and/or sensitivity, there remains the need for an improved method of such detection.
As Mab technology has recently provided researchers with tools to accurately analyze the cell surface components of this bacteria, N. meningtidis proteins are of interest to the epidemiologists as they may provide for a new method of detection as well as a vaccines against it.
One such cell surface protein is the Opacity-Related Protein POPM3 (Stern, 1987, Mol. Microbiol. 1:5-12 incorporated herein by reference). The complete sequence of this 170 amino acid protein is known.
Most meningococcal vaccines have been developed using capsular polysaccharides. One particularly quadravalent vaccine incorporates polyssacharide antigens of serogroups A, C, W and Y, meningococci. However, these serogroups are responsible for less than 49% of memingococcal disease in the United States. No capsular polyssacharide vaccine is available for serogroup B N. meningtidis, which is the most prevalent serogroup, since it is poorly immunogenic. Moreover, polyssacharide vaccines are poorly immunogenic in infants because they are T lymphocyte independent antigens which are inefficient at inducing an immunologic memory. Furthermore, no cross protection between serogroups occurs. Thus, there remains the need for an improved meningococcal vaccine.
It follow then, that there remains a need for at least two products relating to N. meningtidis. The first being a rapid, specific, and sensitive diagnostic test for all strains of N. meningtidis, that does not give false positive results. What is optimally desired is a Mab that will recognize a cell surface antigen that is universally present in most, if not all, strains of N. meningitidis, and, at the same time does not recognize other non-meningitidis causing organisms or material which may be found in conjunction with N. meningtidis. Secondly, it is desirous that the Mab and said protein be used in research towards development of an improved vaccine.
In addition the three major causes of bacterial meningitis, there are other bacterial agents responsible for the disease. One such agent is L. monocytogenes, a motile, grampositive, rod-shaped microorganism belonging to the genus Listeria. This genus is widely distributed in nature-found in soil, water, vegetation and many animal species. See Bille & Doyle, 1990, "Listeria and Erysipelothrix" in Burbert, et al., Manual of Clinical Microbiology 5th ed.:231 which is incorporated herein by reference. Two Listeria species, L. murrayi and L. grayi, are rarely isolated and are presently considered nonpathogenic. However, five other species are genomically related and include three hemolytic species (L. monocytogenes, L. seeligeri and L. ivanovii) and two nonhemolytic species (L. innocua, and L. welshimeri). Of these, only L. monocytogenes, and sometimes L. ivanovii are human pathogens. L. ivanovii is mostly pathogenic for animals (Bille, infra).
Listeria monocytogenes is a facultative intracellular pathogen, capable of growth both in the external environment and inside mammalian cells. It is responsible for opportunistic infections in both humans and animals. The first cases of human listeriosis were reported in the 1930s and outbreaks have been traced to the consumption of contaminated food, most notably dairy and poultry products (Goebel et al., 1991, Infection 19:5195-5197). Individuals at risk are the newborn, the elderly, and the immunocompromised.
Clinical features of the diseases are meningitis and meningoencephalitis. Infection with L. monocytogenes has also been observed as septicemia (with resulting abortion) in pregnant women, and patients with malignancies and immunosuppression. Some people, usually predisposed by an underlying cardiac illness, have been treated for endocarditis resulting from listerial infection.
Although L. monocytogenes is considered an uncommon adult pathogen, it is the third most common cause of bacterial meningitis in neonares (McKay & Lu, 1991, Infection & Immun. 59:4286-4290). Highest mortality and neurological sequelae among survivors is seen when the central nervous system is involved. However, underlying conditions which cause lower cell-mediated immunity, such as transplants, malignancy and AIDS, can result in increased mortality, up to 60%.
There has been a gradual increase in the incidence of human Listeriosis since the 1960s. Presumably, this is related to the increased numbers of individuals with malignancies undergoing radiation and chemotherapy, which allows for their prolonged survival but with immunosuppression as their consequence. Similarly, increases in renal transplantations has exposed increasing numbers of patients to possible infectious complications. Finally, with the rapid spread of AIDS and its suppression of immune function, it can be expected that the occurrence of human listeriosis may increase substantially in the future years.
The epithelial cells of the gastrointestinal tract may be the primary site of entry of L. monocytogenes. It was discovered in the 1960s that this bacterium can invade, survive and replicate within phagocytic cells, such as macrophages and monocytes (Michel & Cossart, 1992, J. Bacteriol. 174:7098-7103 incorporated herein by reference). Nonprofessional phagocytes, which are unable to take up extracelluarly growing bacteria, are also susceptible to invasion by this intracellular organism (Bubert et al., 1992, J. Bacteriol. 174:8166-8171 incorporated herein by reference). Apparently, L. monocytogenes is able to induce its own phagocytosis in these host cells. Specific virulence factors are required for this invasion and intracellular growth.
A major extracellular protein P60, named for its relative molecular weight of 60,000 daltons, is produced by all virulent L. monocytogenes strains. Protein P60 is derived from the Protein P60 Precursor also known as the invasion-associated protein (iap) as described by Koehler, et al., 1990, Infect. Immun. 58:1943-1950 incorporated herein by reference. Moreover, the precursor protein is 484 amino acids in length and the sequence is known.(SEQ ID NO: 26)
Spontaneously occurring mutants of L. monocytogenes that show a decreased level of the protein P60, known as R mutants, are a virulent and unable to invade nonprofessional phagocytes. R mutants are still phagocytized by macrophages with the same efficiency as wild-type bacteria and are able to replicate in these cells. Addition of partially purified P60 protein from wild-type L. monocytogenes restores the invasiveness of these R mutants into nonprofessional phagocytic cells. This finding has led to the conclusion that P60 is involved in the mechanism of uptake of L. monocytogenes by nonprofessional phagocytic cells.
The P60 protein of L. monocytogenes,(SEQ ID NO: 26)is 484 amino acids long, contains a putative N-terminal, signal sequence of 27 amino acids and an extended repeat region of 19 threonine-asparagine units. The middle portion of the protein P60, consisting of about 240 amino acids, and located about 120 amino acids from both the N-and C-terminal ends, varies considerably from the deduced amino acid sequences of the related P60 proteins of L. innocua, L ivanovii, L seeligeri, L welshimeri and L grayi. From the predicted secondary structure and hydropathy studies on this protein, the hydrophillic middle portion consists of two alpha-helical regions flanking the repeat domain. Conversely, the hydrophobic N- and C- terminal ends are in predominantly B-pleated sheets. This would suggest that the middle region is exposed on the protein's surface (Kohler, infra).
The CSF findings in listeria meningitis are quite variable and often result in a negative gram stain. This means that confirmed diagnosis is dependent on culture of either blood or CSF samples, which may take up to 48 hours. Given its high mortality and morbidity, and the increasing numbers of populations at risk, it is apparent that the need exists for rapid diagnosis and for a vaccine against L. monocytogenes infections.
It is a well known feature of bacterial and viral meningitis etiological agents that they possess the ability to infect the CNS. Until recently, it was not known how these agents could pass the Blood-Brain Barrier. The mechanism by which circulating bacteria enter the CSF compartment has only recently been understood. Circulating organisms could invade the CSF compartment by translocation through or between vascular endothelial cells and underlying tissues before entering the CSF. In fact, vascular lesions are a feature of meningitis caused by such organisms as Salmonella choleraesuls and Pasteruella haeloytica. See Wildock, 1977, Vet. Pathol 14:113-120; Sullivan, "The Nervous System: Inflammation", in Jubb et al., eds., 1985, Pathology of Domestic Animals, Volume 1:278-290 all of which are incorporated herein by reference.
However, while vascular endothelial damage may be integral to the pathogenic pathway for some bacteria, it is unlikely to be the mechanism of entry for most cases of meningitis, since vascular lesions are not a prominent early feature of meningitis caused by either N. meningtidis, S. pneumoniae, E coli, S. suls, H. parasuis, H. influenzae, or S. aureus (Williams, 1990, J. Infec. Dis., 162:474-481).
It has been shown that bacteria can be carried into the CSF in association with monocytes migrating into the CSF compartment to maintain populations of resident macrophages (Cordy, 1984, Vet. Pathol 21:593-597). This method of entry for bacteria is also analogous to the mechanism employed by some viruses (HIV, Maaedi-Visna-caprine arthritis encephalitis virus) when invading the CNS. See Peluso, 1985, Virology 147:231-236; Narayan, 1985, Rev. Infec. Dis. 7:899-98; Roy, 1988, J. Leukoc. Clol. 43:91-97; Westervelt, 1991, Vaccines 91:71-76 which are all incorporated herein by reference.
It is also known art that cellular immune reactions consist of a complex series of coordinating events. In response to tissue injury, monocytes are recruited from bone marrow via the blood circulation (Robinson, 1989, PNAS 86:1850-1854 incorporated herein by reference). These activated blood monocytes then differentiate into macrophages in response to several immune mediators produced at the site of inflammation (Yoshimura, et al., 1989, FEBS Letter 244:487-493).
As macrophages normally function to protect the body from potentially toxic substances, either infectious or chemical in nature, they serve as scavengers, processing and presenting antigen to the B lymphocytes, which in turn produce antibodies. (Edington, 1993, Bio/Technology 11:676-681 incorporated herein by reference), Macrophages and also known to secrete mediators that mediate systemic host defence responses and local inflammation.
The first evidence of mediators being involved in cellular immune reactions was noted in 1970 (Ward, 1970, Cell Immunol., 1:162-174). It was reported that addition of antigen to specifically sensitized lymphocytes caused release of an "activity" which attracted macrophages (Robinson, infra). It is now well known that immune mediators possess a variety of functions for cytokines such as the interleukins and interferons.
This led the recent discovery of a family of small, secretory cytokine-like proteins called chemokines for their apparent chemotactic properties, whose complete proinflammatory functions have yet to be elucidated. However, the size and amino acid sequence of many of these chemokines is known as illustrated in Michiel, 1993, Bio/Technology 11:739, incorporated herein in its entirety by reference.
Like most secreted proteins, the chemokines are synthesized with a hydrophobic leader sequence which is cleaved to produce the mature, active chemokine. Comparison of their amino acid sequences has shown these proteins to have a highly conserved pattern of four cysteine residues in the mature peptides. Consequently, they have been classified into two groups based on their structural characteristics: the alpha chemokine group having an intervening amino acids between cys-1 and cys-2, (ie. a C-X-C motif); the beta chemokine group has no intervening amino acid, (ie. a C-C motif). (Michiel, 1993). Cys-1 crosslinks with Cys-3 and Cys-2 crosslinks with Cys-4, resulting in a similar tertiary structure for all the proteins classified into this family of chemokines.
It is further known that the chemokines appear to be functionally involved in cell chemotaxis. Their amino acids sequence diversity suggests that each chemokine has distinct cellular specificity, each having its own unique cellular targets.(Michiel, infra). This cellular specificity appears related to seven transmembrane-domain receptors in each chemokine, but the overlapping pattern of ligand binding and their regulation has yet to be determined (Rollins, et al., 1989, Molecular & Cellular Biol. 9:4687-4695 incorporated herein by reference).
Several peptides from the beta chemokine family have been found to possess the ability for "chemo-attracting" monocytes/macrophages. One such chemotactic protein was identified in 1978 in antigen-stimulated human lymphocytes. (Robinson, infra) and was named LDCF, for Lymphocyte-Derived Chemotactic Factor. This particular chemokine has since been isolated from a variety of different glioma cell lines; human peripheral blood mononuclear leukocytes, (Yoshimura, infra); resting human monocytes (Rollins, infra); human lung fibroblasts and a primary human fetal fibroblast cell line. This latter line being the only member of the Beta family of chemokines to be identified in fibroblasts.
As with all chemokines, various names have been used to identify this protein. The following terms are therefore interchangeable for those skilled in the art: GDCF-2: for Glioma-Derived Monocyte Chemotactic Factor; hJE: for human JE gene product; MCAF: for Monocyte Chemotactic Factor; and MCP: for Monocyte Chemoattractant Protein-1. As the amino acid sequences for these chemokines was found to be identical, the term MCP has been adopted for describing this particular chemokine. It is thus referred to in the art as other chemokines that share significant sequence homology with MCP-1, and have been named MCP-2and MCP-3, according to the order of their discovery.
The amino acid sequence of MCP-1 shows the mature protein to be 99 amino acids long starting at what corresponds to nucleotide 70 of the gene. The functional portion of the protein is known to be the active portion with the first 23 amino acids serving as a signal sequence. MCP-1 is a secretory N-glycosylated glycoprotein of a variety of molecular weights but predominantly occurring at 13,000; 15,000; and 15,500 Daltons with post-translational modification probably accounting for the various forms. The two former isoforms have been named alpha and beta respectively but the structural differences between the two are still unknown. Yet, it is known that their amino acids sequences are identical, apparently derived from a single gene product.
Many mitogenic and activating stimuli appear to cause secretion of MCP-1 by a wide variety of cells. These findings suggest that the cellular regulation of MCP-1 expression is complex, and involves circulating cytokine levels in addition to other factors. Viral and bacterial infections in turn, can affect these levels and are thus involved in the function of MCP-1.
The size and amino acid sequence of MCP-3 is also known as illustrated by Van Damme, et al., 1992, J. Exp. Med. 176:59-65, incorporated herein by reference. It has also been determined that MCP-3 is a chemotactic factor that can attract monocytes and that it can bind heparin.
Amino acids sequences of all proteins described in detail in the present invention are given using the following single letter code: A=ala, C=cys, D=asp, E=glu, F=phe, G=gly, H=his, I=ile, K=lys, L=leu, M=met, N=asn, P=pro, Q=gln, Rh=arg, S=ser, T=thr, V=val, W=trp, Y=tyr.
Accordingly, there remains a significant and urgent need to determine the mechanism used by meningitis etiological agents, as diverse as bacteria and viruses, to attract and infect monocytes and/or gaining access to the CNS. There also remains a significant and urgent need to develop a therapeutic capable of blocking such infection of the CNS by bacterial and viral meningitis etiologic agents utilizing such a mechanism. Specifically, there remains a need in the art for a monoclonal antibody specific for both bacterial and viral infectious agents of meningitis, where said monoclonal antibodies would recognize both bacterial and viral infectious agents of meningitis and have substantial diagnostic utility. Additionally, there is also a need for a known proteinaceous region containing the epitope(s) recognized by said monocional antibody where said epitope or peptide could be chemically synthesized, thereby avoiding the difficulties inherent in purification and administration of larger fragments of the antigenic molecules. An additional need for this said peptide is evident for use in diagnostic test kits to indicate meningitis infection as well as would also be useful in the development of a general meningitis vaccine.